JP2014526259A - プロテアソーム脱ユビキチン化阻害剤スクリーニング - Google Patents
プロテアソーム脱ユビキチン化阻害剤スクリーニング Download PDFInfo
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Abstract
【選択図】 図10
Description
イン ビトロプロテアソーム活性アッセーを、黒96穴マイクロタイタープレート中で、反応バッファ(25mM Hepes,0.5mM EDTA,0.03%SDS)中のヒト20Sプロテアソーム(ボストン ビオケム)を使用して、プロテアソーム活性用の基質としてSuc−LLVY−AMC,Z−LLE−AMC又はBoc−LRRAMCを用いて行った。脱ユビキチナーゼ活性アッセーを、ヒト19S RP(ボストン ビオケム)を用いて、基質としてユビキチン−AMCを用いて行った。異種移植片研究のために、1x106FaDu頭−首細胞又は2x105ルイス肺ガン(LLC)細胞を含む100μlの細胞懸濁液を、SCID又はC578BL/6Jマウスの脇腹に皮下注射した。腫瘍取得後、マウスを対照群又は処理群にランダム化し、そして5mgkg−1のb−AP15又はビヒクルを投与した。イン ビボのレベルのアポトーシス及び細胞死を、M30 Apoptosense(登録商標)及びM65 ELISA(登録商標)アッセー(Peviva)を用いて、血漿中の開裂したカスパーゼの検出及びサイトケラチン−18の合計レベルから決定した。該方法を以下に詳細に記載する。
b−AP15はユビキチン−プロテアソーム系を阻害する。
b−AP15(1μM)で6時間処理したMCF7細胞のCMAP読み取りを表3に示す。
b−AP15は19S RPによる脱ユビキチン化を阻害する。
b−AP15での処理後の19S RP又は26SプロテアソームによるUb−AMC開裂の阻害;一般的DUB阻害剤であるユビキチンアルデヒド(Ubal)を対照として含めた(図2a)。Ub−GFPの19S RP媒介開裂の免疫ブロット(図2b);19S RPをDMSO又は指示した濃度のb−AP15で前処理し、次いで組み換えDUB基質としてUb−GFPを添加した。b−AP15(50μM)処理後の19S RP Ub−GFP開裂の動力学(図2c)。b−AP15はHdm2の脱ユビキチン化を阻害する(図2d);ユビキチン化Hdm2を、DMSO又はb−AP15(50μM)で処理した19S RPに添加し、次いで免疫ブロッティングした。DMSO又はb−AP15(50μM)で処理した後の、K63/K48結合ユビキチン4量体の19S RPによるユビキチン鎖分解反応(図2e)。
b−AP15は19S RP DUBs UCHL5及びUSP14を阻害する。
19S RPをDMSO、NEM(10mM)、b−AP15(50μM)(図3a)又はTPEN(250μM)(図3b)で前処理し、Ub−GFPを添加し、そして抗GFP抗体で免疫ブロッティングした。プロテアソームDUBsの活性部位指向ラベル付け(図3c);精製19S又は26SプロテアソームをDMSO、NEM又はb−AP15で前処理し、次いでHA−UbVSでラベル付けし、そして免疫ブロッティングをした。b−AP15(1μM)で3時間処理したHCT−116細胞の免疫ブロット(図3d);全細胞溶解物からのDUBsをHA−UbVSでラベル付けし、次いでSDS−PAGE及び指示した抗体を用いた免疫ブロッティングを行った。
b−AP15はイン ビボにおける腫瘍の成長を阻害する。
FaDuヒト腫瘍異種移植片を有するSCIDマウスを腫瘍取得(200mm3)について無作為にし、そして毎日、ビヒクル(n=10)又は5mg kg−1のb−AP15(n=15)のいずれかを10日間皮下注射により処理した。平均腫瘍体積±SEMを示す(***P=<0.001)(図4a)。b−AP15処理後の循環する腫瘍誘導CK18及びカスパーゼ開裂(CK18−Asp396)の合計レベル(**p=0.01)(図4b)。HCT−116Bel−2+細胞に挑戦したヌードマウスの無病率(図4c)。マウスをビヒクル(n=6)又は5mg kg−1のb−AP15(n=6)のいずれかで1週当たり4−5回、3週間処理し、そして腫瘍開始について監視した(log−ランク、P=0.0136,ハザード比=7.9)。同系の肺ガン(LLC)腫瘍を有するC57BL/6Jマウスを、ビヒクル(n=4)又は5mg kg−1のb−AP15(n=4)を用いて1日オン/2日オフのサイクルで処理した(図4d);平均腫瘍体積±SEMを示す(P=<0.01)。同所乳癌(4T1)を有するBALB/cマウスを、ビヒクル(n=5)又は2.5mg kg−1のb−AP15(n=5)を用いて1日オン/3日オフのサイクルで処理した(図4e);平均腫瘍体積±SEMを示す(**P=<0.01)。ビヒクル又はb−AP15処理4T1乳癌からの肺転移性コロニーのボックス及びウィスカープロット(図4f);ボックスは上部及び下部の四分位数及び平均を示し、ウィスカーは最大値及び最小値を示す。ビヒクル及びb−AP15処理4T1におけるK48−結合ユビキチン蓄積及び開裂カスパーゼ−3についての代表的な免疫組織化学的染色、元の倍率×20(図4g)。ビヒクル及びb−AP15処理マウスの肝臓及び卵巣におけるAML浸潤(図4h)。ビヒクル処理マウスの肝臓は、グリコーゲン枯渇及び非特異性出血と共に、白血病性芽細胞の浸潤を示した。ビヒクル処理マウスの卵巣は、白血病性芽細胞の大きい浸潤と間質性出血を示した。これと対照的に、b−AP15処理マウスからの肝臓及び卵巣は、浸潤を少ししか示さず、正常な形態を示した(元の倍率×20)。
b−AP15はDNA損傷を誘発しない。
HCT−116細胞をb−AP15又はドキソルビシン(100nM,18時間の遺伝毒性ストレス用の正の対照として)で処理した(図5)。細胞溶解物を、ホスホリル化p53の抗体及びDNA損傷についてのヒストンH2AXマーカーに対して、負荷対照としてのp53及びβ−アクチンの合計レベルで免疫ブロットした。
b−AP15はアポトーシスを誘発しそしてHCT−116細胞の細胞生存を阻害するが、一方PBMC(末梢血液単核細胞)及び不死化hTERT−RPE1は感受性がより低い。
HCT−116細胞を次第に増加する濃度のb−AP15で24時間処理し、アポトーシスのレベルを、ELISAアッセーによりカスパーゼ開裂サイトケラチン−18(CK18)のレベルを測定することにより決定した(図6a)。HCT−116細胞を次第に増加する濃度のb−AP15で48時間処理した。細胞生存率を酸−ホスファターゼ活性アッセーにより決定した。平均値±s.d.を示す(図6b)。HCT−116又はhTERT−RPE1細胞を次第に増加する濃度のb−AP15で72時間処理し、次いでFMCA法(44)を用いて細胞毒性を分析した(図6c)。HCT−116又はhTERT−RPE1細胞を増加する濃度のボルテゾミブで72時間処理し、次いでFMCA法を用いて細胞毒性を分析した(図6d)。HTERT−RPE1は、不死化したヒト網膜色素上皮細胞系である(39)。IC50を、グラフパッドプリズム(グラフパッドソフトウェア社、カルフォルニア、米国)中の対数(log)濃度/効果曲線から、非直線回帰分析(可変ヒル勾配を有する4パラメータモデル)を用いて決定した(図6e,6f)。濃度/反応曲線を、FMCAアッセーを用いて、それぞれ8個の濃度のb−AP15及びボルテゾミブで2倍希釈にて3回生じさせた。結果を4回又は5回の独立した実験から、logIC50+SDとして表す(HCT−116、n=5;PBMC、n=4;hTERT−RPE1、n=5)。
HCT−116細胞の同質遺伝子クローンにおけるアポトーシス誘発の用量反応曲線。
HCT−116細胞を次第に増大する濃度のボルテゾミブ又はb−AP15で24時間処理し、そしてアポトーシスのレベルを、ELISAアッセーによりカスパーゼ開裂サイトケラチン−18(CK−18)のレベルを測定することにより決定した(平均ひだ(折り畳み、fold)変化±s.d.,n=4)(図7)。
b−AP15はプロテアソームのタンパク質分解活性を阻害しない。
20S CP(2nM)をDMSO、b−AP15(50μM)又はボルテゾミブ(100nM)でアッセーバッファ(25mMのHEPES,0.5mMのEDTA,0.03%SDS)中で5分間予備処理し、次いでそれぞれプロテアソームキモトリプシン様、カスパーゼ様及びトリプシン様の活性の分析のために、蛍光性基質(Suc−LLVY−AMC,Z−LLE−AMC又はBoc−LRR−AMC)を100μM添加した(図8a)。アッセーバッファ(25mMのHEPES,50mMのNaCl,1mMのMgCl2,2mMのATP,1mMのDTT)中の26Sプロテアソーム(2nM)を、図8aに示した実験のように処理した(図8b)。値は、ひだ開裂を相対的蛍光単位で表す。
b−AP15は19S及び20S粒子の解離を引き起こさない又はユビキチン結合を変更しない。b−AP15処理プロテアソームの基質オーバーレイアッセー(図9a)。
精製した26Sプロテアソームを、天然ゲル電気泳動法により分離したb−AP15(10μM又は50μM)で処理し、そしてペプチターゼ活性用の蛍光性基質としてSuc−LLVY−AMCを使用してタンパク質分解活性について検定した。ゲルの分析は、対照及びb−AP15レーンの両方で、二重に(RP2CP)及び1重に(RP1CP)蓋されたプロテアソームの存在を示した。0.03%SDSの添加は、蓋されていない20Sコア粒子の存在下で増加を示さなかった。b−AP15はプロテアソーム−ユビキチン結合活性を変更しない(図9b)。HCT−116細胞をボルテゾミブ(100nNM)又はb−AP15(1μM)で処理し、そしてプロテアソームをアフィニティ精製した。会合ポリユビキチンのレベルを免疫ブロット法により決定した。
b−AP15は一般的なDUB阻害剤ではない。
HCT−116をb−AP15(1μM)で3時間処理した(図10)。10mMのN−エチルマレイミド(NEM)で処理した溶解物を全DUB阻害の対照として含ませた。DUB活性を細胞溶解物から、蛍光基質ユビキチン−7−アミド−4−メチルクマリン(Ub−AMC)の開裂を測定することにより決定した。
b−AP15の生化学的特性評価。b−AP15の用量反応(図11a)。
精製した10Sプロテアソーム(5nM)を、指示した濃度のb−AP15で処理し、そしてDUB活性を、Ub−AMC開裂の検出により決定した。IC50値(2.1±0.411μM)を、グラフパッドプリズム中の対数(log)濃度曲線から、非直線回帰分析を用いて決定した(平均値±SD,n=3)。細胞なしアッセーで観察されたIC50は細胞内で観察されたIC50よりも幾分高いことに注目されたい。これは多分、b−AP15の疎水性(XLogP=3.3)により細胞内の該化合物が豊富になることによるであろう(11)。b−AP15阻害の可逆性(図1b)。阻害の可逆性を、酵素/b−AP15複合体の急速な希釈の後、DUB活性の回復を測定することにより決定した。反応に通常使用される19S濃度の50倍(250mM)と、b−AP15についての計算IC50値の10倍(25μM)とを含む反応混合物を氷上で15分培養し、次いで反応バッファ中に50倍希釈して、19Sについては5nMの最終濃度そしてb−AP15については0.5μMの最終濃度を得た。Ub−AMC開裂の線形反応曲線は、b−AP15が可逆的阻害剤であることを示す。b−AP15はシスチン残基と非特異的に反応するか否かの決定(図11c)。19S(5nM)を、還元されたグルタチオン(GSH(2mM))と混合したb−AP15(10μM)又はb−Ap15(10μM)と処理した。グルタチオンの存在は、19S DUB活性のb−AP15媒介阻害を低減しなかった。
b−AP15は一般的なDUB阻害剤ではない。
HCT−116細胞をb−AP15(1μM)で3時間処理し、そしてプロテアソームをアフィニティ精製した(図12a)。プロテアソームDUB活性を、Ub−AMC/suc−LLVY−AMCの開裂として表して、プロテアソーム回復について標準化した(P=0.012、不対t−テスト、2回随行)。b−AP15は非プロテアソーム性DUBを阻害しない(図12b)。組み換え非プロテアソーム性DUBをb−AP15で処理し、%活性を決定した。293T細胞及びHeLa細胞からの細胞溶解物をb−AP15(50μM)で処理し、次いでHA−UbVSを用いて活性ラベル付けした(図12c)。全サンプルをSD−PAGE上で操作し、次いでα−HA抗体を用いて免疫ブロッティングした。
b−AP15処理は動物体重を著しく変更しない(図13)。
対照と異種移植用の処理動物との間で、始点と終点での体重の差異を図4に示した:FaDu、−1.3%;LLC,+2.1%;4T1,+5.8%。ボックスは上部及び下部の四分位数及び平均を示し、ウィスカーは最大値及び最小値を示す。
NC160細胞系内の細胞系のb−AP15及びボルテゾミブに対する感度(図14)。
図示したのは、個々の細胞系についてのIC50値(左側のグラフ)及び各腫瘍型についての中央IC50値(右側のグラフ)である。データはwww.dtp.nci.nih.gov.から取った。矢印は、各薬物に対して2つの最も感度が高い腫瘍細胞型を示す。
b−AP15処理細胞に観察されたシャベロン遺伝子の発現(表1)は、タンパク質毒性反応の誘発を示す。
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Claims (4)
- 化合物が高特異性のプロテアソーム脱ユビキチン化阻害剤か否かを決定するために、該化合物をスクリーニングする方法であって、該化合物を26Sプロテアソームのヒト19S制御粒子(19S RP)と接触させ、そして該化合物が脱ユビキチン化(DUB)酵素UCHL5及びUSP14の活性を阻害するか否かを決定することを含み、ここでUCHL5及びUSP14の活性の阻害は、該化合物が高特異性のプロテアソーム脱ユビキチン化阻害剤であることを示す、上記スクリーニング方法。
- 上記化合物が非プロテアソーム関連DUB酵素の活性に影響を及ぼすか否かを決定する追加のステップを含む、請求項1に記載の方法。
- 上記非プロテアソーム関連DUB酵素が少なくともUCHL1、UCHL3、USP2、USP7及びUSP8から成る群の少なくとも1員を含む、請求項2に記載の方法。
- UCHL5及びUSP14活性の阻害の上記決定は、ユビキチン−AMC又はHA−ユビキチンビニルスルホンを基質としてヒト19S RPを用いて行われるアッセーにより実施される、請求項3に記載の方法。
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